Iron deficiency is still endemic in many parts of the developing world with significant physical and economic consequences. Iron overload seen in association with common hemoglobinpathies such as sickle cell disease and the thalassemias contributes to the pathophysiology of these disease. To properly regulate the absorption of iron in these states requires a detailed knowledge of the mechanisms of intestinal iron transport. Recently, proteins have been described that are involved in the regulation of iron uptake. These proteins include HFE, which when harboring a C282Y mutation causes hemochromatosis, a disease characterized by increased iron absorption. Two iron transporters have been described: NRAMP2, an iron transporter expressed on brush border membrane, and ferroportinl, a basal membrane iron transporter. Hephaestin, a multicopper ferrioxidase and a homologue of cerruloplasm, oxidizes newly absorbed iron, an event required for iron release from intestinal epithelium. Our data has demonstrated that exposure of the small intestine epithelium to iron stimulates the internalization of NRAMP2 from the brush border membrane. We have also demonstrated that NRAMP2 when internalized meets apo-Tf internalized from the basolateral surface in a perinuclear compartment. Based on our data and the work of others, we hypothesize that iron is transported through the intestinal cell via transcytosis. The specific aims of the current proposal are directed to substantiating this hypothesis. In the first speck aim, we will examine the kinetics of NRAMP2 internalization after exposure to iron. In the second specific aim, we will determine if newly absorbed iron and NRAMP2 are in the same vesicles or if NRAMP2 is internalized separtely as a regulatory mechanism. In the third specific aim, we use confocal microscopy to determine if NRAMP2 and apo-Tf, hephaestin, or ferroportin 1 meet and share the same compartment. The fourth specific aim will also use the yeast two-hybrid system to find proteins that interact with NRAMP2. In addition, we will determine the specificity of NRAMP2 internalization and interaction with other proteins for Fe versus other divalent cations.